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Literature summary extracted from

  • Matsumoto, S.; Igura, M.; Nyirenda, J.; Matsumoto, M.; Yuzawa, S.; Noda, N.; Inagaki, F.; Kohda, D.
    Crystal structure of the C-terminal globular domain of oligosaccharyltransferase from Archaeoglobus fulgidus at 1.75 A resolution (2012), Biochemistry, 51, 4157-4166.
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
2.4.99.18 cloning of DNA encoding the full-length AfAglB-S1 gene, expression of N-terminally His6-tagged wild-type and selenomethionine-labeled AglB in Escherichia coli strains BL21(DE3) and C43(DE3), respectively Archaeoglobus fulgidus

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
2.4.99.18 C-terminal globular domain of the of the smallest AglB, purified recombinant wild-type and selenomethionine-labeled AgBs, sitting and hanging drop vapor diffusion methods, crystals grow from a hanging drop with a 1:1 volume ratio of the protein stock solution containing 20 mg/mL protein in 20 mM Tris buffer, pH 8.0, and the reservoir solution containing 0.1 M MES buffer, pH 6.5, and 12.5% PEG 3350, at 20°C, soaking of crystals in reservoir solution with 20% ethylene glycol added for cryoprotection, X-ray diffraction structure determination and analysis at 1.75-1.94 A resolution Archaeoglobus fulgidus

Protein Variants

EC Number Protein Variants Comment Organism
2.4.99.18 D520A site-directed mutagensis of the AglB-S1 subunit of OST, the mutant cannot be expressed in Escherichia coli Archaeoglobus fulgidus
2.4.99.18 D520N site-directed mutagensis of the AglB-S1 subunit of OST, the mutant shows reduced OST activity compared to the wild-type enzyme Archaeoglobus fulgidus
2.4.99.18 D526A site-directed mutagensis of the AglB-S1 subunit of OST, the mutant cannot be expressed in Escherichia coli Archaeoglobus fulgidus
2.4.99.18 E521A site-directed mutagensis of the AglB-S1 subunit of OST, the mutant cannot be expressed in Escherichia coli Archaeoglobus fulgidus
2.4.99.18 E521Q site-directed mutagensis of the AglB-S1 subunit of OST, the mutant shows reduced OST activity compared to the wild-type enzyme Archaeoglobus fulgidus
2.4.99.18 I522A site-directed mutagensis of the AglB-S1 subunit of OST, the mutant shows reduced OST activity compared to the wild-type enzyme Archaeoglobus fulgidus
2.4.99.18 I535A site-directed mutagensis of the AglB-S1 subunit of OST, the mutant cannot be expressed in Escherichia coli Archaeoglobus fulgidus
2.4.99.18 K531A site-directed mutagensis of the AglB-S1 subunit of OST, the mutant shows similar OST activity compared to the wild-type enzyme Archaeoglobus fulgidus
2.4.99.18 K531D site-directed mutagensis of the AglB-S1 subunit of OST, the mutant shows reduced OST activity compared to the wild-type enzyme Archaeoglobus fulgidus
2.4.99.18 K531E site-directed mutagensis of the AglB-S1 subunit of OST, the mutant shows reduced OST activity compared to the wild-type enzyme Archaeoglobus fulgidus
2.4.99.18 K531R site-directed mutagensis of the AglB-S1 subunit of OST, the mutant shows reduced OST activity compared to the wild-type enzyme Archaeoglobus fulgidus
2.4.99.18 M524A site-directed mutagensis of the AglB-S1 subunit of OST, the mutant cannot be expressed in Escherichia coli Archaeoglobus fulgidus
2.4.99.18 N528A site-directed mutagensis of the AglB-S1 subunit of OST, the mutant shows reduced OST activity compared to the wild-type enzyme Archaeoglobus fulgidus
2.4.99.18 T518A site-directed mutagensis of the AglB-S1 subunit of OST, the mutant cannot be expressed in Escherichia coli Archaeoglobus fulgidus
2.4.99.18 T523A site-directed mutagensis of the AglB-S1 subunit of OST, the mutant shows reduced OST activity compared to the wild-type enzyme Archaeoglobus fulgidus

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
2.4.99.18 Mn2+ activates Archaeoglobus fulgidus

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
2.4.99.18 dolichyl diphosphooligosaccharide + [protein]-L-asparagine Archaeoglobus fulgidus
-
 dolichyl diphosphate + [protein]-L-asparagine-N-oligosaccharide a glycoprotein with the oligosaccharide chain attached by N-beta-D-glycosyl linkage to a protein L-asparagine ?
2.4.99.18 dolichyl diphosphooligosaccharide + [protein]-L-asparagine Archaeoglobus fulgidus DSM 4306
-
 dolichyl diphosphate + [protein]-L-asparagine-N-oligosaccharide a glycoprotein with the oligosaccharide chain attached by N-beta-D-glycosyl linkage to a protein L-asparagine ?

Organism

EC Number Organism UniProt Comment Textmining
2.4.99.18 Archaeoglobus fulgidus O29918 NBRC 100126, three AglB paralogues
-
2.4.99.18 Archaeoglobus fulgidus DSM 4306 O29918 NBRC 100126, three AglB paralogues
-

Purification (Commentary)

EC Number Purification (Comment) Organism
2.4.99.18 recombinant N-terminally His6-tagged wild-type and selenomethionine-labeled AglB from Escherichia coli strain BL21 by nickel affinity chromatography and gel filtration, followed by anion exchange chromatography Archaeoglobus fulgidus

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2.4.99.18 dolichyl diphosphooligosaccharide + [protein]-L-asparagine
-
 Archaeoglobus fulgidus dolichyl diphosphate + [protein]-L-asparagine-N-oligosaccharide a glycoprotein with the oligosaccharide chain attached by N-beta-D-glycosyl linkage to a protein L-asparagine ?
2.4.99.18 dolichyl diphosphooligosaccharide + [protein]-L-asparagine
-
 Archaeoglobus fulgidus DSM 4306 dolichyl diphosphate + [protein]-L-asparagine-N-oligosaccharide a glycoprotein with the oligosaccharide chain attached by N-beta-D-glycosyl linkage to a protein L-asparagine ?
2.4.99.18 dolichyl diphosphooligosaccharide + [protein]-L-asparagine i.e. substrate peptide LLO prepared from Pyrococcus furiosus cells and a substrate peptide, TAMRAAla-Pro-Tyr-Asn-Val-Thr-Lys-Arg, which is optimized by peptide library experiments Archaeoglobus fulgidus dolichyl diphosphate + [protein]-L-asparagine-oligosaccharide a glycoprotein with the oligosaccharide chain attached by N-beta-D-glycosyl linkage to a protein L-asparagine, product analysis by mass spectrometry ?
2.4.99.18 dolichyl diphosphooligosaccharide + [protein]-L-asparagine i.e. substrate peptide LLO prepared from Pyrococcus furiosus cells and a substrate peptide, TAMRAAla-Pro-Tyr-Asn-Val-Thr-Lys-Arg, which is optimized by peptide library experiments Archaeoglobus fulgidus DSM 4306 dolichyl diphosphate + [protein]-L-asparagine-oligosaccharide a glycoprotein with the oligosaccharide chain attached by N-beta-D-glycosyl linkage to a protein L-asparagine, product analysis by mass spectrometry ?
2.4.99.18 additional information a set of peptides, TAMRA-Gly-X-X-X-Val-Thr-NH2, where the X-X-X sequence is varied for testing the N-glycosylation dependency, is validated as substrates Archaeoglobus fulgidus ?
-
 ?
2.4.99.18 additional information a set of peptides, TAMRA-Gly-X-X-X-Val-Thr-NH2, where the X-X-X sequence is varied for testing the N-glycosylation dependency, is validated as substrates Archaeoglobus fulgidus DSM 4306 ?
-
 ?

Subunits

EC Number Subunits Comment Organism
2.4.99.18 More Archaeoglobus AglB lacks a beta-barrel-like structure found in other AglBs and PglBs,but AglB contains a kinked helix bearing a conserved motif, called DK/MI motif, it is a variant type of the DK motif with the insertion, this particular type of the DK motif contributes to the activity. Structure comparisons, overview Archaeoglobus fulgidus

Synonyms

EC Number Synonyms Comment Organism
2.4.99.18 oligosaccharyltransferase
-
 Archaeoglobus fulgidus
2.4.99.18 OST
-
 Archaeoglobus fulgidus

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
2.4.99.18 60
-
 assay at Archaeoglobus fulgidus

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
2.4.99.18 7.5
-
 assay at Archaeoglobus fulgidus

General Information

EC Number General Information Comment Organism
2.4.99.18 evolution the catalytic subunit of OST is called STT3 in eukaryotes, AglB in archaea, and PglB in eubacteria. Archaeoglobus f ulgidus encodes three AglB paralogues, two of them are the shortest AglBs across all domains of life. Archaeoglobus fulgidus
2.4.99.18 physiological function oligosaccharyltransferase transfers glycan to asparagine in the N-glycosylation sequon, the lysine and isoleucine residues in the DK/MI motif participate in the Ser/Thr recognition in the sequon Archaeoglobus fulgidus